Cellular Uptake of Chloroquine Is Dependent on Binding to Ferriprotoporphyrin IX and Is Independent of NHE Activity in Plasmodium falciparum (original) (raw)
Related papers
Biochemical Pharmacology, 1986
Chloroquine accumulation by human erythrocytes infected with nine different strains of the malarial parasite Plasmodium falciparum, which varied by greater than or equal to 20-fold sensitivity to the drug, was measured as a function of time and drug concentration. Although the kinetics of uptake were clearly quite complex in this system, at least two general phases were observed, an extremely rapid short phase (less than 30 sec), followed by a slower phase leading to steady state within 60 min. The concentration of chloroquine in the parasite food vacuole quickly exceeded 1 mM at 10(-6) M external drug concentration. Minor alkalinization of this organelle was observed during the first 30 sec; this pH was reduced progressively over time in a concentration-dependent manner. The rate of pH reduction was highest in the drug-sensitive strains. Neither the rate of chloroquine accumulation nor intracellular chloroquine concentrations at steady state could adequately differentiate sensitive from resistant strains. Higher intracellular drug concentrations were required to kill resistant versus sensitive strains, suggesting that a change in sensitivity to chloroquine of an intracellular effector is the mechanism of resistance. The rapid rate and extensive accumulation of chloroquine, and the lack of significant alkalinization, indicate that a new theory of the mechanism of antimalarial action of the drug is required.
Antimicrobial Agents and Chemotherapy, 2007
New antimalarials are urgently needed. We have shown that tetrahydroquinoline (THQ) protein farnesyltransferase (PFT) inhibitors (PFTIs) are effective against the Plasmodium falciparum PFT and are effective at killing P. falciparum in vitro. Previously described THQ PFTIs had limitations of poor oral bioavailability and rapid clearance from the circulation of rodents. In this paper, we validate both the Caco-2 cell permeability model for predicting THQ intestinal absorption and the in vitro liver microsome model for predicting THQ clearance in vivo. Incremental improvements in efficacy, oral absorption, and clearance rate were monitored by in vitro tests; and these tests were followed up with in vivo absorption, distribution, metabolism, and excretion studies. One compound, PB-93, achieved cure when it was given orally to P. berghei-infected rats every 8 h for a total of 72 h. However, PB-93 was rapidly cleared, and dosing every 12 h failed to cure the rats. Thus, the in vivo results corroborate the in vitro pharmacodynamics and demonstrate that 72 h of continuous high-level exposure to PFTIs is necessary to kill plasmodia. The metabolism of PB-93 was demonstrated by a novel technique that relied on double labeling with a radiolabel and heavy isotopes combined with radiometric liquid chromatography and mass spectrometry. The major liver microsome metabolite of PB-93 has the PFT Znbinding N-methyl-imidazole removed; this metabolite is inactive in blocking PFT function. By solving the X-ray crystal structure of PB-93 bound to rat PFT, a model of PB-93 bound to malarial PFT was constructed. This model suggests areas of the THQ PFTIs that can be modified to retain efficacy and protect the Zn-binding N-methyl-imidazole from dealkylation. . † Supplemental material for this article may be found at http://aac .asm.org/.
Accumulation of chloroquine by membrane preparations from Plasmodium falciparum
Molecular and Biochemical Parasitology, 1990
Chloroquine susceptibility and resistance have been associated respectively with the uptake and efflux of chloroquine by Plasmodiumfalciparum. We made membrane preparations from parasitized and unparasitized red cells in order to study chloroquine accumulation in a cell-free system. The accumulation of [3H]chloroquine by these preparations is inhibited by unlabeled chloroquine and thus is specific. Only membranes from parasitized red cells demonstrate time-dependent chloroquine accumulation; membranes from unparasitized red cells do not. Chloroquine accumulation is eliminated by detergent (0.05% Triton X-100) and reduced by a hypertonic medium, consistent with accumulation inside membrane vesicles rather than binding to membranes. Accumulation is energy dependent; it has a specific requirement for ATP, which cannot be replaced with GTP, CTP, UTP, T'I'P or ADP, an apparent Km of 21 /JM and an apparent Vma~ of 4.6 pmol (mg protein)-j h-j. Vesicle acidification is MgATP dependent, and is reversed by NI-I4CI. Chloroquine accumulation is inhibited by reduced medium pH, N-ethylmaleimide or oligomycin, but not by vanadate or ouabain. These studies demonstrate that membrane vesicles prepared from parasitized red cells provide a model system for the study of chloroquine accumulation by P. falciparum.
PLoS ONE, 2012
Haemoglobin degradation during the erythrocytic life stages is the major function of the food vacuole (FV) of Plasmodium falciparum and the target of several anti-malarial drugs that interfere with this metabolic pathway, killing the parasite. Two multi-spanning food vacuole membrane proteins are known, the multidrug resistance protein 1 (PfMDR1) and Chloroquine Resistance Transporter (PfCRT). Both modulate resistance to drugs that act in the food vacuole. To investigate the formation and behaviour of the food vacuole membrane we have generated inducible GFP fusions of chloroquine sensitive and resistant forms of the PfCRT protein. The inducible expression system allowed us to follow newly-induced fusion proteins, and corroborated a previous report of a direct trafficking route from the ER/Golgi to the food vacuole membrane. These parasites also allowed the definition of a food vacuole compartment in ring stage parasites well before haemozoin crystals were apparent, as well as the elucidation of secondary PfCRT-labelled compartments adjacent to the food vacuole in late stage parasites. We demonstrated that in addition to previously demonstrated Brefeldin A sensitivity, the trafficking of PfCRT is disrupted by Dynasore, a non competitive inhibitor of dynamin-mediated vesicle formation. Chloroquine sensitivity was not altered in parasites over-expressing chloroquine resistant or sensitive forms of the PfCRT fused to GFP, suggesting that the PfCRT does not mediate chloroquine transport as a GFP fusion protein. Citation: Ehlgen F, Pham JS, de Koning-Ward T, Cowman AF, Ralph SA (2012) Investigation of the Plasmodium falciparum Food Vacuole through Inducible Expression of the Chloroquine Resistance Transporter (PfCRT). PLoS ONE 7(6): e38781.
Access to Hematin: the Basis of Chloroquine Resistance
Molecular pharmacology, 1998
The saturable uptake of chloroquine by parasites of Plasmodium falciparum has been attributed to specific carrier-mediated transport of chloroquine. It is suggested that chloroquine is transported in exchange for protons by the parasite membrane Na ϩ /H ϩ exchanger [J Biol Chem 272:2652-2658 (1997)]. Once inside the parasite, it is proposed that chloroquine inhibits the polymerization of hematin, allowing this toxic hemoglobin metabolite to accumulate and kill the cell [Pharmacol Ther 57:203-235 (1993)]. To date, the contribution of these proposed mechanisms to the uptake and antimalarial activity of chloroquine has not been assessed. Using sodium-free medium, we demonstrate that chloroquine is not directly exchanged for protons by the plasmodial Na ϩ /H ϩ exchanger. Furthermore, we show that saturable chloroquine uptake at equilibrium is due solely to the binding of chloroquine to hematin rather than active uptake: using Ro 40-4388, a potent and specific inhibitor of hemoglobin digestion and, by implication, hematin release, we demonstrate a concentration-dependent reduction in the number of chloroquine binding sites. An
Biochemical Pharmacology, 2007
Chloroquine (CQ), a 4-aminoquinoline, accumulates in acid ic digestive vacuoles of the malaria parasite, preventing conversion of toxic haematin to B-haematin. We examine how bis 4-aminoquinoline piperaquine (PQ) and its hydroxy-modification (OH-PQ) retain potency on chloroquine-resistant (CQ-R) Plasmodium falciparum. For CQ, PQ, OH-PQ and 4 and 5, representing halves of PQ, B-haematin inhib itory activity (BHIA) was assayed, while potency was determined in CQ-sensitive (CQ-S) and CQ-R P. falciparum. From measured pKas and the pH-modulated distribution of base between water and lipid (logD), the vacuolar accumulation ratio (VAR) of charged drug from plasma water (pH7.4) into vacuolar water (pH4.8) and lip id accumulation ratio (LAR) were calculated. All agents were active in BHIA. In CQ-S, PQ, OH-PQ and CQ were equally potent while 4 and 5 were 100 times less potent. CQ with 2 basic centres has a VAR of 143,482, while 4 and 5, with 2 basic centres of lower pKas have VARs of 1,287 and 1,966. In contrast PQ and OH-PQ have 4 basic centres and achieve VARs of 104,378 and 19,874. This confirms the importance of VAR for potency against CQ-S parasites. Contrasting results were seen in CQ-R. 5, PQ and OHPQ with LARs of 693; 973,492 and 398,118 (compared with 8.25 for CQ) showed similar potency in CQ-S and CQ-R. Importance of LAR for potency against CQ-R parasites probably reflects ability to block efflux by hydrophobic interaction with PfCRT but may relate to B-haematin inhibition in vacuolar lip id.
Antimicrobial Agents and Chemotherapy, 2009
Polymorphisms in the Plasmodium falciparum crt (Pfcrt), Pfmdr1, and Pfmrp genes were not significantly associated with quinine (QN) 50% inhibitory concentrations (IC 50 s) in 23 strains of Plasmodium falciparum. An increased number of DNNND repeats in Pfnhe-1 microsatellite ms4760 was associated with an increased IC 50 of QN (P ؍ 0.0007). Strains with only one DNNND repeat were more susceptible to QN (mean IC 50 of 154 nM). Strains with two DNNND repeats had intermediate susceptibility to QN (mean IC 50 of 548 nM). Strains with three DNNND repeats had reduced susceptibility to QN (mean IC 50 of 764 nM). Increased numbers of NHNDNHNNDDD repeats were associated with a decreased IC 50 of QN (P ؍ 0.0020). Strains with profile 7 for Pfnhe-1 ms4760 (ms4760-7) were significantly associated with reduced QN susceptibility (mean IC 50 of 764 nM). The determination of DNNND and NHNDNHNNDDD repeats in Pfnhe-1 ms4760 could be a good marker of QN resistance and provide an attractive surveillance method to monitor temporal trends in P. falciparum susceptibility to QN. The validity of the markers should be further supported by analyzing more isolates.
Antimicrobial Agents and Chemotherapy, 2007
New antimalarials are urgently needed. We have shown that tetrahydroquinoline (THQ) protein farnesyltransferase (PFT) inhibitors (PFTIs) are effective against the Plasmodium falciparum PFT and are effective at killing P. falciparum in vitro. Previously described THQ PFTIs had limitations of poor oral bioavailability and rapid clearance from the circulation of rodents. In this paper, we validate both the Caco-2 cell permeability model for predicting THQ intestinal absorption and the in vitro liver microsome model for predicting THQ clearance in vivo. Incremental improvements in efficacy, oral absorption, and clearance rate were monitored by in vitro tests; and these tests were followed up with in vivo absorption, distribution, metabolism, and excretion studies. One compound, PB-93, achieved cure when it was given orally to P. berghei-infected rats every 8 h for a total of 72 h. However, PB-93 was rapidly cleared, and dosing every 12 h failed to cure the rats. Thus, the in vivo results corroborate the in vitro pharmacodynamics and demonstrate that 72 h of continuous high-level exposure to PFTIs is necessary to kill plasmodia. The metabolism of PB-93 was demonstrated by a novel technique that relied on double labeling with a radiolabel and heavy isotopes combined with radiometric liquid chromatography and mass spectrometry. The major liver microsome metabolite of PB-93 has the PFT Znbinding N-methyl-imidazole removed; this metabolite is inactive in blocking PFT function. By solving the X-ray crystal structure of PB-93 bound to rat PFT, a model of PB-93 bound to malarial PFT was constructed. This model suggests areas of the THQ PFTIs that can be modified to retain efficacy and protect the Zn-binding N-methyl-imidazole from dealkylation. by guest http://aac.asm.org/ Downloaded from oping world because the developing world bears most of the morbidity and the mortality burden. Drugs for the developing world must be inexpensive and easily administered. The product profile of an antimalarial drug useful for the developing world includes oral bioavailability, a maximum 3 days of therapy for cure, and once-or twice-daily dosing (17). The in vitro pharmacodynamics of THQ PFTIs demonstrated that 3 days of exposure at levels 10 to 50 times the concentration that led to 50% growth inhibition (the 50% effective dose [ED 50 ]) was necessary for the complete killing of P. falciparum (16). As noted above, the initial THQ compounds had issues with poor oral bioavailability and rapid clearance. Thus, for THQ PFTIs to become useful as antimalarials, compounds with improved oral absorption and reduced clearance must be found.